Central to Q fever pathogenesis is replication of the causative agent, Coxiella burnetii, in a large and spacious phagolysosome-like Coxiella-containing vacuole (CCV). Recruitment of membrane during CCV biogenesis is a complex process modulated by both host and bacterial factors. Coxiella encodes a specialized Dot/Icm type IVB secretion system (T4BSS) that secretes proteins with effector functions directly into the host cell cytosol. Effector proteins are predicted to modulate an array of host cell processes, such as vesicular trafficking, that promote pathogen growth. By using new gene inactivation technologies developed in our laboratory, we have confirmed that a functional T4BSS is required for productive infection of human macrophages by Coxiella. Furthermore, we have verified Dot/Icm-dependent secretion by different strains of Coxiella of over 50 proteins. Coxiella must co-opt vesicular trafficking pathways to promote PV development. We are currently elucidating the activities of five effector proteins that traffic to the PV membrane termed CvpA (Coxiella vacuolar protein A), CvpB, CvpC, CvpD, and CvpE that are speculated to modulate membrane fusion events. Mutants in individual cvp genes all display significant defects in replication and PV development. Particular insight into the function of CvpA has been gained by showing the protein subverts clathrin-coated vesicle trafficking. CCV formation involves interactions with autophagosomes. We examined that autophagy regulator mTOR activity in response to Coxiella infection to better understand how the pathogen regulates lysosomal physiology to promote CCV biogenesis. Infected THP-1 cells and primary human macrophages exhibited reduced phosphorylation of the mTOR substrate 4E-BP1. Infected cells also displayed impaired mTORC1 reactivation and lysosomal relocalization when transitioned from amino acid-deprived to nutrient-rich conditions. Inhibition of mTOR was T4BSS-dependent, and cells infected with Coxiella and cultured under mTOR-inhibiting conditions supported larger and more fusogenic CCVs. Hyperactivation of mTOR inhibited Coxiella growth. Infected cells did not exhibit altered autophagic flux under any condition tested. However, during prolonged amino acid deprivation, infected cells accumulated LC3 and p62. Based on these data, inhibition of mTOR is predicted to alter normal lysosomal physiology to generate the expansive Coxiella CCV. Regulation of the Coxiella T4BSS is poorly defined. IcmS is a predicted cytoplasmic adapter protein that facilitates translocation of certain T4BSS effectors by binding an internal signal sequence(s). We examined the function of Coxiella IcmS by generating an icmS deletion mutant. The Coxiella icmS mutant grows normally in axenic media while having a pronounced growth defect in host cells that is rescued with a single chromosomal copy of icmS. Optimal secretion of individual substrates is either IcmS-dependent or independent. Additionally, a subset of substrates display hyper-secretion in the Coxiella icmS mutant, suggesting IcmS may also suppress secretion of some Dot/Icm substrates. Thus, regulation by IcmS appears complex with the growth defect of the Coxiella icmS mutant potentially explained by both deficient and aberrant secretion of effector proteins. A hallmark of Coxiella is a biphasic developmental cycle that generates biologically, ultrastructurally, and compositionally distinct large cell variant (LCV) and small cell variant (SCV) forms. LCV are replicating, exponential phase forms while SCVs are non-replicating, stationary phase forms. The SCV has several properties, such as a condensed nucleoid and an unusual cell envelope, suspected of conferring enhanced environmental stability. Although the developmental cycle is considered fundamental to Coxiella virulence, the molecular biology of this process is poorly understood. Ultrastructural studies show marked differences in the cell envelope between cell variants, but little is known about biochemical differences between SCV and LCV that confer their distinct biological and physical properties. We analyzed the lipid composition of Coxiella after 4 (LCV), 7 (intermediate forms) and 14 (SCV) days of growth in synthetic medium, using thin layer chromatography and mass spectrometry. Similar to Escherichia coli, Coxiella contains cardiolipin, phosphatidylglycerol (PG), and phosphatidylethanolamine (PE). PE and PG are present in lower quantities in the SCV relative to the LCV. Interestingly, the SCV contains substantial amounts free fatty acids, which are normally toxic to bacteria. Mutational analysis indicates that SCV-enriched lipids are generated via the activity of a Coxiella outer membrane phospholipase A (CBU0489). A cbu0489 mutant exhibits a significant growth defect in THP-1 macrophage-like cells, suggesting developmentally regulated lipid synthesis is required for optimal intracellular growth and could contribute to the distinct properties of LCV and SCV. To further identify genetic determinants of LCV to SCV transition, we profiled the Coxiella transcriptome by microarray at 3 (early LCV), 5 (late LCV), 7 (intermediate forms), 14 (early SCV) and 21 (late SCV) days post-infection (dpi) of Vero epithelial cells. A striking transcriptional signature of the SCV is induction (10-fold) of five genes encoding predicted L,D transpeptidases that catalyze beta-lactam resistant 3-3 peptide crosslinks typically found in the peptidoglycan (PG) of stationary phase bacteria. In addition to increased 3-3 crosslinking of muropeptide stems in SCV PG, mass spectrometry revealed glycine residues and additional modifications of peptide stems that may confer unique cell wall properties Using an innovative and sensitive shotgun proteomics approach, we further analyzed PG of Coxiella SCVs and discovered a new mechanism of outer membrane (OM) stabilization involving covalent linkage of PG to OM porins. PG muropeptides are linked to the N-terminal glycine residue of Coxiella OmpA-like porins CBU0307 and CBU0311. Deletion of Coxiella ldt2, encoding L,D transpeptidase 2, abolishes glycine linkages. Striking phenotypes of the deltaldt2 mutant are pronounced membrane blebbing and production of outer membrane vesicles. This hitherto unrecognized mechanism of PG-OM anchoring dramatically expands our understanding of OM stabilization and the function of L,D transpeptidases. These findings have important implications for understanding how OM permeability is controlled to allow entry of small molecules, such as antibiotics. Moreover, it invokes a new model of OM stabilization in bacteria lacking PG-linked Brauns lipoprotein.